Sickle cell disease is a major clinical problem in the US, France, the United Kingdom, Brazil, and in Africa, India and the Middle East. A recent study shows that the annual cost of medical care in the US for people who suffer from sickle cell disease exceeds $1.1 billion. Sickle hemoglobin (HbS) polymerization drives the pathophysiology of this disease. Fetal hemoglobin (HbF) is present throughout fetal development and is replaced by adult hemoglobin (HbA). A major therapeutic goal in sickle cell disease is to induce high HbF levels in sickle erythrocytes. We contend that the amount of HbF per F-cell (HbF/F-cell) and the distribution of concentrations of HbF/F-cells is a critical determinant of disease severity. Existing measurement of hemolysate HbF concentration or the number of F-cells in blood does not measure HbF/F-cell or the distribution of HbF/F- cell in a patient blood sample. This unmet need is critical because even patients with high HbF can have severe disease since their HbF is unevenly distributed among F-cells and many F-cells have insufficient HbF concentrations to inhibit HbS polymerization. We hypothesize that the proportion of F-cells that have enough HbF to thwart HbS polymerization is the most critical predictor of the likelihood of severe sickle cell disease. HbF/F-cell should be the phenotype studied in HbF induction therapeutics and would also be useful in developing risk scores that project likelihoods for disease severity. A clinical method for rapidly ascertaining HbF/F-cell is currently not available. As HbF concentrations among F-cells are highly variable and clinically relevant, the predictive value of HbF would be improved with an assay measuring the distribution of HbF concentrations within F-cells. Physicians would be able to provide better care using an HbF diagnostic as a standard method to assess which patients have higher risk, as well those patients with lower risk. Our goal in this application is to develop a prototype diagnostic test that will then be developed as laboratory developed test (LDTs) in a Clinical Laboratory Improvement Amendments (CLIA) laboratory in a subsequent Phase 2 SBIR study, based on the pilot data obtained in this study. The specific aims of this proposal are to first develop, optimize and validate an HbF/F-cell diagnostic and then to evaluate the diagnostic in a pilot study of human subjects with sickle cell disease. The long-term goal is to develop a test based on a flow cytometry kit for HbF/F-cell profiling and a license for use of an online tool for disease risk determination that can be done in any hospital with flow cytometry and internet capability.